The macula is in the center of the retina, directly behind the lens in the eye. It is a tiny area with an yellow color consisting of xanthophylls like lutein, (R,R)-zeaxanthin and (R,S)-zeaxanthin and hence called macular xanthophylls. These act as antioxidants protecting the retina from oxidative degradation and help in sharp vision needed to read, write, drive and see objects clearly. A life time slow and steady damage of the macula can lead to age related macular degeneration (AMD) and cataract. The macular xanthophylls in the diet or supplementation can help in maintaining healthy eyes. Lutein and (R,R)-zeaxanthin can be derived from fruits and vegetables while (R,S)-zeaxanthin from sea foods or dietary supplements or from bio conversion of lutein within the body. Of the various classes of the pigments, the carotenoids are among the most widely distributed in nature with red, yellow and orange color having varied functions like light harvesting and protection against destructive photo-oxidation in terrestrial plants.
Although specific carotenoids have been identified in various fruits and vegetables, bird feathers, egg-yolk, poultry skin, crustaceans and macular eye region, they are especially abundant in marigold petals, corn and leafy vegetables. The correlation between dietary carotenoids and carotenoids found in human serum and plasma indicate that only selected groups of carotenoids make their entry into the human blood stream to exert their effect. Each carotenoid shows an individual pattern of absorption, plasma transport and metabolism.
Carotenoids absorb light in the 400-500 nm region of the visible spectrum. This physical property imparts the characteristic yellow/red colour to the pigments. Carotenoids contain a conjugated backbone composed of isoprene units, which are usually inverted at the center of the molecule, imparting symmetry. Changes in geometrical configuration about the double bonds result in the existence of many cis- and trans-isomers. Mammalian species do not synthesize carotenoids and therefore these have to be obtained from dietary sources such as fruits, vegetables and egg yolks. In the recent years, carotenoids have been reported to have several health benefits, which include prevention and or protection against serious health disorders.
Carotenoids are non-polar compounds classified into two sub-classes, namely, polar compounds called xanthophylls or oxy-carotenoids and non-polar hydrocarbon carotenes like beta-carotene, lycopene, etc. Both the sub-classes have at least nine conjugated double bonds responsible for the characteristic colors of the carotenoids. Xanthophylls have ring structures at the end of the conjugated double bond chain with polar functions like hydroxyl or keto group. The examples for xanthophylls include lutein, zeaxanthin, capsanthin, canthaxanthin, beta-cryptoxanthin, astaxanthin, etc. As natural colorants and also for their role in human health, xanthophylls like lutein, (R,R)-zeaxanthin and (R,S)-zeaxanthin have attracted the renewed attention of scientists and researchers in the biomedical, chemical and nutritional field in recent years.
Lutein and zeaxanthin contribute to yellow and orange-yellow colors respectively. Lutein and zeaxanthin can be present in plant material in the free form and also in ester form. Lutein is present in green leafy vegetables like spinach, kale and broccoli in the free form; fruits like mango, orange and papaya; red paprika, algae, yellow corn, contain lutein in the form of its esters. It is also present in the blood stream and various tissues in human body and particularly in the macula, lens and retina of the eye.
Lutein is chemically designated as beta-ε-carotene 3,3′-diol. Zeaxanthin is formed by the addition of two hydroxy groups to beta-carotene. Since the hydroxy positions are in 3 and 3′-, the chemical name for zeaxanthin is beta, beta-carotene-3,3′-diol. The common name of zeaxanthin is derived from Zea mays because this carotenoid was first identified in corn (Zea mays).
It can be seen below that lutein is not symmetrical as the position of double bond in the left ring is not identical with the double bond position in the right ring. Zeaxanthin is completely symmetrical with regards to left and right rings due to an extra conjugated double bond compared to lutein.
Xanthophylls can show both optical (R- and S-stereo isomers) and geometrical isomers (trans, E- and cis, Z-). The conformation of R- and S-stereo isomers is based on CD spectral and chiral column HPLC studies while the conformation of cis- and trans-isomers is based on electronic, infrared, NMR, HPLC-MS and HPLC-NMR on-line spectroscopy studies. It is well known that when an organic molecule has a carbon atom with four different types of atoms or groups attached to it, that carbon atom is designated as chiral carbon atom. The chiral carbon atom is responsible for two different spatial arrangements leading to the formation of optical isomers while the number of double bonds of the polyene chain and the presence of a methyl group and the absence of steric hindrance decide the number of trans- and cis-isomers. In the case of trans-zeaxanthin, the carbon atoms at 3 and 3′ positions in the two end rings are both chiral atoms.
Thus, trans-zeaxanthin has two chiral centers at the carbon atoms C3 and C3′, based on the positions of the secondary hydroxy groups attached to them. Therefore, there are four possible stereo isomers of trans-zeaxanthin namely, (3R-3′R)-isomer, (3S-3′S)-isomer and (3R-3′S)- or (3S-3′R)-isomer. In these isomers (3R-3′S)- and (3S-3′R)- are identical. Thus, there are three chiral isomers of trans-zeaxanthin. The isomer causing rotation of polarized light in a right handed manner is called R-stereo isomer, the isomer causing left handed rotation. S-stereo isomer and the third isomer possessing a two fold opposite effects (R,S; optically inactive) which is called meso-form of zeaxanthin. The structural formulae of lutein, (R,R)-zeaxanthin and (R,S)-meso zeaxanthin are given below.

Chemical Structures of Macular Xanthophylls
The conjugated double bonds of lutein and zeaxanthin contribute to the distinctive colors of each pigment, and also influence the ability of these to quench singlet oxygen. Due to the extra conjugated double bond, zeaxanthin is believed to be a stronger anti-oxidant compared to lutein.
The macular pigment of the eye is composed primarily of three xanthophyll pigments (or macular xanthophylls), namely (3R,3′R,6′R)-lutein, (3R,3′R)-zeaxanthin and (3R,3′S)-zeaxanthin in the order 36, 18 and 18% of the total carotenoid content of the retina along with the remaining 20% consisting of minor carotenoids like oxo-lutein, epi-lutein and ε-,-ε-carotene 3,3′-dione (J. T. Landrum and R. A. Bone, Lutein, Zeaxanthin and the Macular Pigment, Arch. Biochem. Biophys., 385, 28-40, 2001).
Although these xanthophyll pigments are found throughout the tissues of the eye, the highest concentration is seen in the macula lutea region of the retina, including a central depression in the retina called fovea. The concentration of xanthophyll pigments increases progressively towards the center of the macula and in the fovea, the concentration of these xanthophyll pigments are approximately thousand fold higher than in other human tissues. (Landrum et al., Analysis of Zeaxanthin Distribution within Individual Human Retinas, Methods in Enzymology, L. Packer (editor) 213A, 457-467, Academic Press 1992). The fovea is a relatively small area within the macula, in which the cone photoreceptors reach their maximal concentration. About 50% of the total amounts of the xanthophylls are concentrated in the macula where zeaxanthin dominates over lutein by ratio of 2:1 (Handelman et al., Measurements of Carotenoids in Human and Monkey Retinas, in Methods in Enzymology, L. Packer (editor) 213A, 220-230, Academic Press, NY, 1992; Billsten et al., Photophysical Properties of Xanthophylls in Carotene Proteins from Human Retina, Photochemistry and Photobiology, 78, 138-145, 2003). At the center of the retinal fovea, zeaxanthin is 50:50 mixture of (trans-3R,3′R)-zeaxanthin and (trans-3R,3′S)-zeaxanthin along with small quantity of (3S,3′S)-zeaxanthin (J. T. Landrum and R. A. Bone, Lutein, Zeaxanthin and The Macular Pigment, Arch. Biochem., Biophy., 385, 28-40, 2001).
The fovea is particularly important for proper visual function (eg, acuity) disease and damage to this area is known to result in legal blindness. For example, age-related macular degeneration (AMD) is characterized by pathological changes in the retina, retinal pigment epithelium (RPE) and/or the choroids and preferentially affects the macular region of the retina. This is the leading cause of irreversible vision loss in the United States among those more than 65 years old and there is no established treatment available for most patients. The loss of central vision results in the possible inability to recognize faces, to read or write or drive a car and therefore has a significant effect on an individual's ability to live independently. There is ample epidemiological evidence which supports a role for dietary intake of lutein and zeaxanthin in different isomeric forms in protection against age-related cataract and macular degeneration. The detection of oxidation products of lutein and zeaxanthin in the human retina supports the hypothesis that dietary lutein and zeaxanthin may act as antioxidants in the macular region. (Khachik et al., Identification of Lutein and Zeaxanthin Oxidation Products in Human and Monkey Retinas, Invest. Opthalmol. and Vis. Sci., 38, 1802-1811, 1997)
Of the 40 to 50 carotenoids typically consumed in the human diet, lutein and zeaxanthin, are deposited at an up to 5 fold higher content in the macular region of the retina as compared to the peripheral retina. Zeaxanthin is preferentially accumulated in the foveal region, whereas lutein is abundant in the perifoveal region.
Regarding the location of xanthophylls at a cellular level, they are reported to be bound to specific proteins referred to as xanthophylls binding protein (XBP). The XBP is suggested to be involved in the uptake of lutein and zeaxanthin from the blood stream and stabilization of the same in the retina. The study of xanthophylls and XBP by femto-second transient absorption spectroscopy showed better stability for (3R,3′S)-zeaxanthin enriched XBP compared to (3R,3′R)-zeaxanthin while the photo physical properties of the xanthophylls: (3R,3′R)-zeaxanthin and (3R,3′S,meso)-zeaxanthin are generally identical. It is likely that the meso-zeaxanthin is better accommodated with XBP wherein the protein protects the xanthophylls from degradation by free radicals. Thus, the complex may be a better antioxidant than the free xanthophylls, facilitating improved protection of ocular tissue from oxidative damages. (Billsten et al., Photophysical Properties of Xanthophylls in Caroteno proteins from Human Retina, Photochemistry and Photobiology, 78, 138-145, 2003)
Several functions have been attributed to macular pigments including the reduction of the damaging effects of photo-oxidation from blue light absorbed by the eye, reduction of the effects of light scatter and chromatic aberration on visual performance, and protection against the adverse effects of photochemical reactions because of the antioxidant properties of the carotenoids.
The ability to increase the amount of macular pigment by dietary supplementation with lutein has been demonstrated (Landrum et al., Dietary Lutein Supplementation Increases Macular Pigment, FASEB. J, 10, A242, 1996). The reduced vision function due to cataract and the adult blindness due to AMD can be substantially controlled by consuming fruits and vegetables and dietary supplements containing lutein and (R,R)-zeaxanthin and (R,S)-zeaxanthin available from sea foods denying the vegetarian population. Although (R,S)-zeaxanthin present in eye is considered a metabolic product originating from lutein, the need for dietary supplementation of (R,S)-zeaxanthin is now recognized to improve the macular pigment density. (Landrum and Bone, Functional Foods and Nutraceuticals, 1 Sep. 2001). Similarly, the study has shown that (R,R)-zeaxanthin gains entry to blood and finally to macula. (Breithaupt et al., Comparison of Plasma Responses in Human subjects after the Ingestion of (3R,3′R)-zeaxanthin Dipalmitate from Wolfberry (Lycium barbarum) and Non-esterified (3R,3′R)-zeaxanthin using Chiral HPLC, Brit. J. Nutr. 91, 707-713, 2004). Lutein and zeaxanthin dietary supplements in human trials have shown to raise the macular pigment density and serum concentrations of these carotenoids (Bone et. al., Lutein and Zeaxanthin Dietary Supplements Raise Macular Pigment Density and Serum Concentrations of These Carotenoids in Humans, J. Nutr., 133, 992-998, 2003).
Dietary Sources of Lutein and Zeaxanthin
Lutein is a common carotenoid found in most fruits and vegetables, while zeaxanthin in the (R,R)-isomer form is present only in minute quantities in most fruits and vegetables. Dietary sources of zeaxanthin are limited to greens, certain yellow/orange fruits and vegetables such as corn, nectarines, oranges, papaya, persimmons and squash. Capsicum annum is another most common spice widely used which is a good source of zeaxanthin. Wolfberry (Lycium barbarum), fructus lycii or Gou Qi Zi plant has small red berries which are commonly used in Chinese home cooking and has been shown to have a high content of zeaxanthin (mainly as zeaxanthin dipalmitate) but negligible amounts of lutein. The dried fruit of wolfberry is prescribed by Chinese herbalists as a therapeutic agent for a number of eye diseases. In France, lutein dipalmitate (Helenien) isolated from the blossom leafs of Helenium autumnale is reported to be used for the treatment of the visual disorders. (Wolfgang Gau, Hans-Jurgen Ploschke and Christian Wunsche, Mass Spectrometric Identification of Xanthophylls Fatty Acid Esters From Marigold Flowers (Tagetes erecta) Obtained by HPLC and Craig Counter Current Distribution, J. Chrom. 262,277-284, 1983)
As already mentioned earlier, the dietary source of meso-zeaxanthin is mainly from seafoods like shrimps, fish, turtle, etc, thereby the vegetarian population is deprived of meso-zeaxanthin. However, there is a patent available for pharmaceutical composition containing meso-zeaxanthin for the treatment of retinal disorders like increasing the deposition of macular pigments in the human eye and therapeutic treatment or prophylaxis of AMD (Howard et al., Meso-zeaxanthin Formulations for Treatment of Retinal Disorders, U.S. Pat. No. 6,329,432, 2001).
Lutein and zeaxanthin occur naturally in trans-isomeric form in fruits, vegetables and flowers (marigold). Because of processing conditions due to heat and light, a small percentage of trans- is converted into cis-isomeric form. Therefore, the preferred bio-available form is trans-isomeric as evidenced from the data of geometric isomers compositional analysis of human plasma. (Khachik et al., Isolation and Structure Elucidation of Geometric Isomers of Lutein, Zeaxanthin in Extracts of Human Plasma, J. Chrom. 582, 153-156, 1992). In view of this, it is desirable to use the trans-isomeric form of lutein and zeaxanthin as (R,R)-, (R,S)- in dietary supplements.
To date, little is known about the mechanism of formation, uptake and deposition of meso-zeaxanthin in the retina of the eye. Khachik et al. have reported the presence of 2-3% of (3R,3′S,meso)-zeaxanthin in twenty normal human plasma samples and proposed the metabolic pathways of its formation from dietary lutein and zeaxanthin. It is not clear whether the deposition of meso-zeaxanthin in the retina routes through serum or are produced from lutein/zeaxanthin within the retina. (Khachik et al., in a chapter on Dietary carotenoids and their metabolites as potentially useful chemo protective agents against cancer, in “Antioxidant food supplement in human health, Eds. Packer et al., Academic Press London, page 203-29, 1999). However, Breithaupt et al. did not find the presence of meso-zeaxanthin in human plasma obtained 24 hrs after ingestion of (3R,3′R)-zeaxanthin (ester or free form) in a single blind cross over study using two groups each consisting of six volunteers. The chiral LC-ApcI-MS was used for detection in the pooled plasma sample. (Breithaupt et al., Comparison of plasma responses in human subjects after the ingestion of 3R,3′R-zeaxanthin Dipalmitate from Wolfberry (Lycium barbarum) and Non-esterified 3R,3′R-zeaxanthin using Chiral HPLC, Brit. J. Nutr. 91, 707-713, 2004).
There is evidence and reasons supporting the hypothesis that the carotenoids lutein, zeaxanthin and meso-zeaxanthin are readily bio-available and consequently increase macular pigment levels (Landrum and Bone, Meso-zeaxanthin—A Cutting Edge Carotenoid, Functional Foods and Nutraceuticals, 10 Sep. 2001; Bone et al. Macular Pigment Response to a Supplement Containing Meso-zeaxanthin, Nutr. Metabol. 11.1-8 (2007); Bone et al., Macular Pigment Response to a Xanthophyll Supplement of Lutein, Zeaxanthin and Meso-zeaxanthin. Proc. Nutr. Soc., 105A, (2006); Thurnham et al., Macular Zeaxanthin and Lutein—a Review of Dietary Sources and Bio-availability and Some Relationship with Macular Pigment Optical Density and Age-related Macular Disease, Nutr. Res. Reviews, 20, 163-179 (2007); Thurnham et al., A Supplementation Study in Human Subjects with a Combination of meso-zeaxanthin, (3R,3′R)-Zeaxanthin and (3R,3′R,6′R)-Lutein, Brit. J. Nutr. 99, 1-8, 2008); E. E. Connolly et al., Augmentation of macular pigment following supplementation with all three carotenoids: An exploratory study” Current Eye Res., 35, 335-351 (2010).
In present days, there is high demand for xanthophyll crystals containing high amounts of trans-lutein and/or zeaxanthin for its use as antioxidants, prevention of cataract and macular degeneration, as lung cancer-preventive agent, as agents for the absorption of harmful ultra-violet light from sun rays and quencher of photo-induced free radical and reactive oxygen species, etc. A number of commercial products from natural source are now available to facilitate the formulation of industrial and commercial products with lutein or (R,R)-zeaxanthin. However, to our knowledge xanthophyll composition containing all the essential macular xanthophylls and high concentrations of particularly trans-lutein at least 85% and balance comprising of (R,R)-zeaxanthin and (R,S)-zeaxanthin in equal or higher ratios derived from the same natural source (marigold flower petals) as commercial lutein or zeaxanthin are not available. Evidence of the protective role of trans-lutein, (R,R)-zeaxanthin and (R,S)-zeaxanthin in maintaining eye health has been found based on correlation between dietary supplements vs serum levels and the macular pigment density (Bone et al, Macular Pigment Response to a Supplement Containing Meso-zeaxanthin, Lutein and Zeaxanthin, Nutr. Metabol. 11.1-8 (2007); Bone et al., Macular Pigment Response to a Xanthophyll Supplement of Lutein, Zeaxanthin and Meso-zeaxanthin. Proc. Nutr. Soc., 105A, (2006); Thurnham et al., A Supplementation Study in Human Subjects with a Combination of Meso-zeaxanthin, (3R,3′R)-zeaxanthin and (3R,3′R,6′R)lutein, Brit. J. Nutr. 99, 1-8 (2008).